Glycolysis and respiration are independently regulated in the cell, and their interaction is critical for active muscle. Glycolysis provides the majority of substrate for respiration (i.e., oxidative phosphorylation) in exercise. However, glycolysis can also limit oxidative phosphorylation by generating H+ and lactate under aerobic conditions which reduces intracellular pH and inhibits mitochondrial respiration. The PI's working hypothesis is that the interaction of glycolysis with respiration limits muscle respiration in exercise to well below the mitochondrial capacity. Newly developed magnetic resonance methods are applied to characterize the contractile, oxidative and glycolytic fluxes during in vivo exercise. Human and animal muscle groups differing in these metabolic properties will be used to evaluate the interaction of these pathways. Specific Aim 1 determines the limit to oxidative phosphorylation in exercise. Activation of respiration prior to the onset of glycolysis is used to elicit higher oxidative phosphorylation rates than measured under steady state conditions. The range of metabolic properties among muscles is used to determine how the interaction of these pathways limits oxidative phosphorylation. Specific Aim 2 uses physiological methods to reduce or enhance glycolytic acidification during exercise. These manipulations of intracellular pH are used to determine the role of H+ accumulation in limiting oxidative phosphorylation. Specific Aim 3 determines how altering the ratio of glycolytic to oxidative capacities in a muscle affects the limit to oxidative phosphorylation during exercise. Endurance training is used to increase oxidative capacity vs. glycolytic flux, while sprint training is used to enhance glycolytic relative to oxidative flux. The results may demonstrate and quantify important ways by which glycolytic flux can limit oxygen consumption in skeletal muscle. This project has clinical relevance because many muscle disorders show functional deficits and reflect poor integration of metabolic systems, e.g., diabetes. An understanding of the integration of metabolism in healthy tissue is the first step to understanding how failure of integration limits function in diseased muscle.